Protective Effect Of Melatonin On Cochlear Hair Cell Damage Induced By Nicotine

Objective: Smoking is one of the major risk factors affecting human health worldwide.In addition to its effects on tumors,smoking is associated with many other pathological changes,including cardiovascular,lung and brain diseases.Hearing loss is a common public and social health problem associated with both intrinsic and extrinsic factors.Recent epidemiological studies have shown that smoking can increase the risk of hearing loss,and the risk tends to be higher during years of heavy smoking.Deciphering the mechanisms of smoking-induced hearing loss could therefore be of great value in improving the quality of life of smokers and non-smokers exposed to second-hand smoke.Nicotine,one of the main components in tobacco smoke,is obviously addictive and can circulate rapidly to various tissues in the body.Animal and human studies examining the effects of nicotine have shown a variety of health hazards.Including nervous,cardiovascular,respiratory,kidney,digestive and other systems.Further research is needed to determine whether nicotine poses a risk of hearing damage.Melatonin is a well-known anti-inflammatory and antioxidant that plays a vital role in a variety of physiological functions.Clinically,melatonin has a good therapeutic effect on tinnitus.However,it is not clear whether melatonin has a protective effect on smoking-related hearing loss.In this study,we established a cell model of cochlear hair cell injury induced by nicotine in vitro and explored its related mechanisms.In addition,we evaluated the protective effect of melatonin on this cellular model,providing scientific evidence for the use of melatonin in smoking-related hearing loss.Methods:1.Cultured cochlear hair cells(HEI-OC1)were stimulated with nicotine solution to establish an in vitro cell model of cochlear hair cell injury induced by nicotine.MTT colorimetric assay was used to detect the effects of nicotine on cell activity.The effective concentration of nicotine on he I-OC1 was selected and the 50% inhibitory concentration(IC50)of nicotine on HEI-OC1 was calculated.Flow cytometry and TUNEL were used to detect the apoptosis induced by nicotine.Western blot was used to detect the expression of Bcl-2,Bax,caspase-3/cleaved-capase-3,and PARP/cleavedPARP in cochlear hair cells(HEI-OC1)induced by nicotine.2.To study the mechanism of nicotine-induced injury of cochlear hair cells(HEI-OC1)in vitro.The expression of inflammatory cytokines TNF-α,IL-6 and IL-1β in cochlear hair cells(HEI-OC1)induced by nicotine was detected by enzymelinked immunosorbent assay(ELISA).The nuclear migration of NF-KB P65 was detected after cochlear hair cells(HEI-OC1)were induced by Immunofluorescence.The expressions of COX-2,P-P65,CHOP and Bip in cochlear hair cells(HEI-OC1)induced by nicotine were detected by Western blot.Oxidative stress of cochlear hair cells(HEI-OC1)induced by nicotine was detected by reactive oxygen species(ROS)assay kit.3.The protective effect of melatonin was observed by treating nicotine-stimulated cochlear hair cells(HEI-OC1)with melatonin.The effects of different concentrations of melatonin on nicotineinduced damage of cochlear hair cells(HEI-OC1)were detected by MTT colorimetry.The effective concentration of melatonin was selected to inhibit nicotine-induced cochlear hair cell damage(HEI-OC1).Flow cytometry and TUNEL were used to detect the apoptosis of cochlear hair cells(HEI-OC1)stimulated by nicotine treated with melatonin.Western blot was used to detect the expression of Bcl-2,Bax,caspase-3/ cleaved-capese-3,PARP/cleaved-PARP,CHOP,and Bip after melatonin treatment on nicotine-stimulated cochlear hair cells(HEI-OC1).The expression of TNF-α,IL-6 and IL-1β in nicotine-stimulated cochlear hair cells(HEI-OC1)was determined by enzymelinked immunosorbent assay(ELISA).Reactive oxygen species(ROS)assay kit was used to detect the oxidative stress level of melatonin on nicotine-stimulated cochlear hair cell(HEI-OC1).Results:1.The toxicity of nicotine to HEI-OC1 cochlear hair cells was dose-dependent(0,2.5,5,10,20,40 and 80 μM).Nicotine(10 μM)stimulated he I-OC1 cell apoptosis significantly.Nicotine increases cleaved-caspase-3,Bax and cleaved-ARP expression in cohlear hair cells(HEI-OC1),inhibits bcl-2 expression,promotes HEI-OC1 cochlear hair cells apoptosis.2.Nicotine promotes the expression of pro-inflammatory cytokines TNF-α,IL-6,IL-1β and COX2.Positive regulation of NF-κB signaling Road,thereby aggravating cochlear hair cell inflammation,causing cochlear hair cell damage.Nicotine exposure induces the accumulation of ROS in cochlear hair cells,leading to oxidative stress and injury and apoptosis of cochlear hair cells.Nicotine induced apoptosis of cochlear hair cells induced by ER stress by increasing the expression of ER stress-related proteins CHOP and Bip.3.Melatonin dose-dependently reduced the cytotoxicity of nicotine-induced HEI-OC1 cells(0,25,50,and 100 μM).Further studies showed that melatonin(100 μM)could effectively reduce the apoptosis of HEI-OC1 cells induced by nicotine.Melatonin alleviates nicotine-induced cochlear hair cells by reducing the inflammatory response of cochlear hair cells,reducing the accumulation of reactive oxygen species,and inhibiting endoplasmic reticulum stress.Conclusion: Nicotine promotes cochlear hair cell death by increasing inflammation,ROS,and endoplasmic reticulum stress.In addition,melatonin effectively alleviates nicotine-induced cochlear hair cell damage,suggesting that melatonin may be an effective drug for the treatment of hearing loss caused by smoking.